Aspergillus strains such as Aspergillus sojae and Aspergillus oryzae have been industrially used in the production of brewed food such as soy sauce, sake (rice wine), soybean paste, etc. With a recent determination of the whole genomic sequence of Aspergillus oryzae and development of an exhaustive analysis of gene expression using a micro-array, it has been expected that genetic modification of their genes, especially their chromosomal modification would increase the productivity of an enzyme and improve a growth rate of these filamentous fungi.
Furthermore, enzymes produced by the Aspergillus strains are utilized in a variety of industries. For example, in the production of soy sauce, the traditional food in Japan, various enzymes produced by the Aspergillus strains are used. The Aspergillus strains are cultured with starting materials such as soybean and wheat so as to produce the various enzymes, which will degrade proteins, carbohydrates, lipids, etc. of the soybean and wheat and promote lactic acid fermentation and yeast fermentation in the following steps. If a large amount of the above degradative enzymes is produced, the productivity can be much improved due to the increase in a utility factor of the starting materials and a rate of squeezing. In addition to that, since substrates shall be sufficiently provided to the lactic acid fermentation and yeast fermentation so as to properly carry out the fermentation processes, the quality of soy sauce can be improved to a large extent.
Unlike Aspergillus nidulans, niger, fumigatus and awamori that have a mononuclear generation, koji molds such as Aspergillus sojae and Aspergillus oryzae are always kept in a multinuclear state in their whole life cycle including in a conidium condition, and their sexual generation has not yet been observed. Their nuclear-distribution mechanism from a parent cell to a daughter cell has not yet been revealed, either. Accordingly, a mutant cannot be produced by means of mating between strains or RIP (Repeat Induced Mutation), which makes it difficult to study their genetics. As a result, the genetic analysis of Aspergillus sojae and Aspergillus oryzae has fallen behind in spite of their industrially very high utility.
As it is therefore very important from an industrial point of view to breed Aspergillus strains of high utility such as those having high productivity of various enzymes, methods for breeding have been vigorously developed for the above purpose. There are two prominent types of such breeding methods, i.e., mutation and genetic recombination (genetic modification) methods.
The mutation method uses various mutation treatments such as X-ray, ultraviolet ray, and heavy ion beam. Useful characteristics have been used as an index for screening strains having various enzyme activities and excellent fermentation properties. Recently, the strains having such useful characteristics have been analyzed by means of genomic information to reveal that the duplication of chromosome is important for providing the Aspergillus strains with such useful characteristics. An Aspergillus strain having duplication of such a large scale as 900 kb or more was obtained by means of mutation treatment method (Patent Literature 1). It was already reported that repeated sequences were found with a high frequency at a boundary region of the duplicated region on the chromosome by analysis of a yeast obtained by means of γ-ray radiation (Non Patent Literature 1).
However, the mechanism of such duplication of the chromosome has not yet been revealed. Accordingly, it has been just possible to accidentally obtain a strain having duplication of a region relating to an enzyme by means of conventional mutation treatments and screening based on activity of said enzyme. However, since mutation will actually occur on various sites at random on the chromosome, it has been impossible to duplicate a particular region on the chromosome of the Aspergillus strains. Furthermore, it is well known that back mutation or revertant (elimination of the duplicated chromosome) will occur with a high frequency due to recombination between the homologous sequences in said strain having the duplication obtained by means of the conventional mutation treatments.
On the other hand, the genetic recombination method utilizes transformation to introduce a target gene into the Aspergillus strains for breeding. Conventionally, the gene that can be introduced in said method has 5˜6 kb. However, in order to actually obtain such useful Aspergillus strains as those having a high enzyme-productivity, it would be necessary to include a promoter region, a terminator region and a marker gene for screening, if necessary, in addition to the target gene region in a region to be introduced, which could enlarge the size of said region up to 10 kb or more.
As seen form the above description, there has exited no technique that enables the duplication of a relatively large region at an arbitrary site on the chromosome.